Method for the production of sialylated oligosaccharides

ABSTRACT

Disclosed are methods for the enzymatic synthesis of α-sialylated oligosaccharide glycosides. Specifically, in the disclosed methods, α2,3-sialyltransferase is used to transfer an analogue of sialic acid, employed as its CMP-nucleotide derivative, to the non-reducing sugar terminus of an oligosaccharide having a fucosyl group in the penultimate saccharide unit to the non-reducing sugar terminus. The analogue of sialic acid and the oligosacchairde employed in this method are selected to be compatible with the sialyltransferase employed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/146,285, filed on Sep. 3, 1998 now U.S. Pat. No. 6,194,178 B1, whichis incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This application is directed to methods for the enzymatic synthesis ofα-sialylated oligosaccharides. Specifically, in the methods of thisinvention α2,3-sialyltransferase is employed to transfer sialic acid oran analogue thereof, employed as its CMP-nucleotide, to the non-reducingterminus of an oligosaccharide which oligosaccharide has a fucosyl groupin the position penultimate to the non-reducing sugar terminus of theoligosaccharide.

2. References

The following references are cited in this application as superscriptnumbers at the relevant portion of the application and are incorporatedherein in their entirety.

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3. State of the Art

Carbohydrates and/or oligosaccharides are present on a variety ofnatural and pathological glycoconjugates¹. Of particular interest arecarbohydrates and oligosaccharides containing sialic acid residuesparticularly at the nonreducing sugar terminus³¹. Such sialic acidterminated carbohydrates and oligosaccharides are present in a number ofproducts which have been implicated in a wide range of biologicalphenomena based, in part, on the concept of recognition signals carriedby the carbohydrate structures and by their binding to specific ligands.

Specifically, such sialic acid terminated carbohydrates andoligosaccharides are believed to be receptors for the binding oftoxins⁴, pathogenic agents such as viruses⁵, and are believed to berecognition sites for a variety of lectins, particularly those involvedin cellular adhesion^(6,7), etc.

Similarly, certain oligosaccharides including sialic acid terminatedoligosaccharides have been identified as capable of suppressing acell-mediated immune response to an antigen. The ability of sucholigosaccharides to suppress a cell mediated immune response to anantigen is described by Venot et al.³

Additionally, the presence of certain sialyl terminated oligosaccharidesin tumor-related antigens is documented in the art¹ and, in general, thestructures of the oligosaccharides present on such antigens have beenmodified in some way from normal oligosaccharides so as to lead to theexpression of tumor related antigens². The prospect of passiveimmunotherapy with monoclonal antibodies directed against somesialylated tumor-associated antigens, such as the gangliosides GD₂, GD₃and GM₂, in patients with melanoma has been investigated^(8,9).

The synthesis of such oligosaccharides often involves complex chemicalreactions with corresponding low yields. Accordingly, there has beenmuch interest in using glycosyltransferases in synthesizing at least apart of these molecules.

Glycosyltransferases are a highly polymorphic group of membrane-boundenzymes of endoplasmic reticulum and Golgi bodies that catalyze thetransfer of a single monosaccharide unit from a nucleotide donor to thehydroxyl group of an acceptor saccharide in the biosynthesis of N-glycan(Asn-GlcNAc N-glycosidic linkage; GlcNAc, N-acetylglucosamine) andO-glycan (Ser/Thr-GalNAc, O-glycosidic linkage; GalNAc,N-acetylgalactosamine) moieties of glycoproteins and glycolipids.

The eukaryotic sialyltransferases comprise a family ofglycosyltransferases that catalyze the transfer of N-acetylneuraminicacid (NeuAc), a sialic acid (SA), from CMP-SA to the non-reducingterminus of oligosaccharide chains of glycoconjugates. The addition ofthe sialic acid normally terminates oligosaccharide chain elongationexcept for polysialic chains found on neural cell adhesion molecule andgangliosides.

Known eukaryotic sialyltransferases involved in the synthesis of N- andO-glycan derivatives of the glycoprotien and glycolipid are summarizedin Table 1, dapted from Palcic⁶³. In the table, the R represents theremainder of the acceptor glycoprotein, glycolipid or oligosaccharidechain.

TABLE 1 sialyltransferase EC (SL) Number Linkage Synthesized Gal(2-6)-ST2.4.99.1 NeuAcα2→6Galβ1→4GlcNAc-R (ST6N) GalNACα(2-6)- 2.4.99.4NeuACα2→6GalNAcα-R ST (ST6OI) Gal(2-3)-ST 2.4.99.4NeuACα2→3Galβ1→4GalNAcα-R (ST3O) Gal(2-3)-ST 2.4.99.6NeuAcα2→3Galβ1→3/4GlcNAc-R (ST3N) GalNAcα(2-6)- ST (ST6OII) 2.4.99.7

N-Ac-neuramide 2.4.99.8 NeuAcα2→8NeuAcα2→Galβ-R α(2-8)- sialytransferaseGalβ1-3GlcNAc- NeuAcα2→3Galβ1→3GlcNAc-R ST

α2,3-sialyltransferases are useful eukaryotic enzymes for in vitrosynthesis of N-linked and O-linked sialyl derivatives of glycoproteins,for determinations of acceptors, and other qualitative and quantitativeresearch of glycoproteins. However, it was previously reported that2,3-sialyltransferases would not synthesize N-linked and O-linked sialylderivatives of glycoproteins or glycolipids where the acceptorglycoprotein or glycolipid possessed a fucosyl derivative in thepenultimate position to the non-reducing sugar terminus of theoligosaccharide (U.S. Pat. No. 5,374,655⁶⁷). This necessitated carefulplanning in the synthesis of certain fucosylated and sialylatedoligosaccharides and in some cases required that certain steps becompleted using chemical synthesis, rather than enzymatic synthesis.

In view of the above, it would be particularly advantageous to developmethods for the facile preparation of α-sialylated oligosaccharides fromoligosaccharides having a fucosyl derivative in the penultimate positionto the non-reducing sugar terminus of the oligosaccharide. The presentinvention accomplishes this by using an α2,3-sialyltransferase to effectefficient coupling of sialic acid activated as its CMP-nucleotidederivative (a donor saccharide) to a saccharide or an oligosaccharidehaving a fucosyl derivative in the penultimate position of thenon-reducing end of the sugar moiety (acceptor oligosaccharide).

SUMMARY OF THE INVENTION

The present invention is directed to methods for the synthesis ofoligosaccharides, glycoproteins and glycolipids terminated in thenon-reducing sugar end by an analogue of N-acetylneuraminic acid. Inparticular, the methods of this invention employ α2,3-sialyltransferasesto transfer a sialic acid or analogue thereof, activated as theirCMP-nucleotide derivatives, to the non-reducing terminus ofoligosaccharide acceptors.

Accordingly, in one of its method aspects, the present invention isdirected to a method for the enzymatic synthesis of an α-sialylated andfucosylated oligosaccharide containing a sialic acid or analogue thereofwhich method comprises the steps of:

a) selecting a sialyltransferase compatible with a fucosylatedoligosaccharide having a fucose group in the non-reducing penultimatesaccharide position;

b) selecting a CMP-sialic acid or an analogue thereof which iscompatible with the sialyltransferase selected in step (a);

c) contacting said CMP-sialic acid or an analogue thereof with afucosylated oligosaccharide of the formula

wherein R₁ represents a saccharide residue, R₂ represents a saccharideresidue, and R₁ and R₂ together represent an acceptor for the selectedsialyltransferase; n is from 0 to about 10, Y is selected from the groupconsisting of O, NH and S, and R₃ is selected from the group consistingof H, a protein, a lipid or an aglycon moiety having at least one carbonatom, in the presence of the sialyltransferase selected in step (a)above under conditions whereby the sialic acid or analogue thereof istransferred from the CMP-sialic acid or analogue thereof to thenon-reducing sugar terminus of the fucosylated oligosaccharide so as toform an α-sialylated fucosylated oligosaccharide containing a sialicacid or analogue thereof.

This invention is also directed to a method for the enzymatic synthesisof a fucosylated and α-sialylated oligosaccharide which method comprisesthe steps of:

a) selecting a sialyltransferase capable of sialylating anoligosaccharide having a fucose group in the non-reducing penultimatesaccharide position;

b) selecting a fucosyltransferase;

c) selecting a CMP-sialic acid or an analogue thereof which iscompatible with the sialyltransferase selected in step (a);

d) selecting a GDP-fucose or an analogue thereof which is compatiblewith the fucosyltransferase selected in step (b);

e) contacting said CMP-sialic acid or an analogue thereof and saidGDP-fucose or an analogue thereof with an oligosaccharide of the formula

 R₁—R₂-(saccharide)_(n)-Y—R₃

wherein R₁ represents a saccharide residue, R₂ represents a saccharideresidue, and R₁ and R₂ together represent an acceptor for the selectedsialyltransferase and the selected fucosyltransferase; n is from 0 toabout 10, Y is selected from the group consisting of O, NH and S, and R₃is selected from the group consisting of H, a protein, a lipid or anaglycon moiety having at least one carbon atom, in the presence of saidsialyltransferase and said fucosyltransferase selected in (a) and (b)above, under conditions whereby the sialic acid or analogue thereof andthe fucose or analogue thereof are transferred from the CMP-sialic acidor analogue thereof and the GDP-fucose or analogue thereof,respectively, to the non-reducing sugar terminus of the oligosaccharideso as to form an α-sialylated fucosylated oligosaccharide.

This invention is also directed to a method for determining thenon-reducing terminus structure of an unknown oligosaccharide acceptor,which method comprises the steps of:

a) contacting the oligosaccharide acceptor with a sialyltransferasewhich is not capable of sialylating a non-reducing terminus of anoligosacchairde having a fucose group in the non-reducing penultimatesaccharide position and determining whether the oligosaccharide wassialylated;

b) contacting the oligosaccharide with a sialyltransferase which iscapable of sialylating a non-reducing terminus of an oligosacchairdehaving a fucose group in the non-reducing penultimate saccharideposition and determining whether the oligosaccharide was sialylated; and

c) comparing the results to determine whether the non-reducing terminuswas fucosylated. If the first aliquot of the unknown acceptor was notsialylated in step (a) and the second aliquot of acceptor was sialylatedin step (b) then the acceptor was fucosylated in the non-reducingpenultimate saccharide position.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the discovery that certainα2,3-sialyltransferases will transfer compatible analogues of sialicacid to certain oligosaccharides, glycoproteins, and glycolipids havinga fucosyl group in the penultimate position to the non-reducing end ofthe sugar. This discovery permits the synthesis of oligosaccharidesα-sialylated at the non-reducing terminus from oligosaccharides having afucosyl group in the penultimate position to the non-reducing end of thesugar. This method also permits the transfer of compatible analogues ofsialic acid to the fucosylated oligosaccharide. This invention alsopermits the determination of the structure of acceptors and otherqualitative and quantitive research of glycoproteins and glycolipids.

However, prior to discussing this invention in further detail, thefollowing terms will first be defined.

A. Definitions

As used herein, the following terms have the definitions given below:

The term “sialic acid” means all of the naturally occurring structuresof sialic acid including5-acetoamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonic acid(“Neu5Ac”) and the naturally occurring analogues of Neu5Ac, includingN-glycolyl neuraminic acid (Neu5Gc) and 9-O-acetyl neuraminic acid(Neu5,9Ac₂), which are compatible with the selected sialyltransferase. Acomplete list of naturally occurring sialic acids known to date areprovided by Schauer³¹.

Naturally occurring sialic acids which are recognized by a particularα2,3-sialyltransferase so as to bind to the enzyme and are thenavailable for transfer to an appropriate acceptor oligosaccharidestructure are said to be compatible with the sialyltransferase and aresometimes referred to herein as a “compatible naturally occurring sialicacid”.

The term “analogues of sialic acid” refers to analogues of naturallyoccurring structures of sialic acid including those wherein the sialicacid unit has been chemically modified so as to introduce, modify and/orremove one or more functionalities from such structures. For example,such modification can result in the removal of an OH functionality, theintroduction of an amine functionality, the introduction of a halofunctionality, and the like. In so far as the sialic acid analogues arecompatible with the sialyltransferase, they are sometimes referred toherein as a “compatible sialic acid analogues”.

Certain analogues of sialic acid are known in the art and include, byway of example, 9-azido-Neu5Ac, 9-amino-Neu5Ac, 9-deoxy-Neu5Ac,9-fluoro-Neu5Ac, 9-bromo-Neu5Ac, 8-deoxy-Neu5Ac, 8-epi-Neu5Ac,7-deoxy-Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac,4-N-acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, 3-hydroxy-Neu5Ac,3-fluoro-Neu5Ac acid as well as the 6-thio analogues of Neu5Ac. Thenomenclature employed herein in describing analogues of sialic acid isas set forth by Reuter et al.²⁰

Insofar as sialyltransferases are designed to transfer or donatecompatible naturally occurring sialic acids, analogues of Neu5Ac aresometimes referred to herein as “artificial donors” whereas thecompatible naturally occurring sialic acids are sometimes referred toherein as the “natural donors”.

The term “sialyltransferase” refers to those enzymes which transfer acompatible naturally occurring sialic acid, activated as its cytidinemonophosphate (CMP) derivative, to the terminal oligosaccharidestructures of glycolipids or glycoproteins (collectivelyglycoconjugates) and include enzymes produced from microorganismsgenetically modified so as to incorporate and express all or part of thesialyltransferase gene obtained from another source, including mammaliansources. Numerous sialyltransferases have been identified in theliterature with the different sialyltransferases generally beingdistinguished from each other by the terminal saccharide units on theglycoconjugates which accept the transferase.⁶⁴ For example,sialyltransferases, which build the following terminal oligosaccharidestructures on glycoconjugates have been characterized:

αNeu5Ac(2-3)βGal(1→3/4)βGlcNAc-²¹

αNeu5Ac(2-6)βGal(1-4)βGlcNAc-^(21, 22)

αNeu5Ac(2-3)βGal(1-3)αGalNAc-²³-25

αNeu5Ac(2-6)αGalNAc-²⁶-28

αNeu5Ac(2-6)βGlcNAc-^(29, 30).

Other sialyltransferases with a variety of specificities have beenisolated from a variety of sources.

A “sialyltransferase compatible with a fucosylated oligosaccharidehaving a fucose group in the non-reducing penultimate saccharideposition” means that the sialyltransferase is capable of sialylating anoligosaccharide having a fucose group or analogue thereof in thenon-reducing penultimate saccharide position. It has been found that themyxoma virus α2,3-sialyltransferase, as disclosed in InternationalPatent Application Publication No. WO97/18302⁶⁵, has this capability.

It is contemplated that related sialyltransferases also encoded by othergenera of the sub-families of Chorodopoxvirinae, Entomopoxvirinae andthe unclassified viruses of the family of Poxviridae will be compatiblewith a fucosylated oligosaccharide.

Analogues of sialic acid activated as their cytidinemonophosphatederivative which are recognized by a particular sialyltransferase so asto bind to the enzyme and are then available for transfer to anappropriate acceptor oligosaccharide structure are said to be compatiblewith the sialyltransferase and are sometimes referred to herein as a“compatible analogue of sialic acid”. Because the transfer reactionemploys a sialyltransferase, it goes without saying that an analogue ofsialic acid employed in such a reaction must be a compatible analogue ofsialic acid.

CMP-nucleotide derivative of Neu5Ac refers to the compound:

CMP-derivatives of analogues of sialic acid refer to those compoundshaving structures similar to that above with the exception that theNeu5Ac residue is replace with an analogue of sialic acid.

The term “fucosyltransferase” refers to those enzymes which transfer acompatible naturally occurring fucose, activated as its guanosinediphosphate (GDP) derivative, to the terminal oligosaccharide structuresof glycolipids or glycoproteins (collectively glycoconjugates) andinclude enzymes produced from microorganisms genetically modified so asto incorporate and express all or part of the fucosyltransferase geneobtained from another source, including mammalian sources. Numerousfucosyltransferases have been identified in the literature.

The term “analogues of fucose” refers to analogues of naturallyoccurring structures of fucose including those wherein the fucose unithas been chemically modified so as to introduce, modify and/or removeone or more functionalities from such structures. For example, suchmodification can result in the removal of an OH functionality, theintroduction of an amine functionality, the introduction of a halofunctionality, the introduction of a sulfate or phosphate moiety, andthe like. Certain analogues of fucose are known in the art and include,by way of example, 3-deoxy-fucose⁶⁸, arabinose, C-6 modified fucoses⁶⁹(i.e. 6-O-propyl fucose) and 3,6 dideoxy-L-galactose⁷⁰.

It is also contemplated that the fucose or analogues of fucose may betransferred from other purine diphosphates including,adenosine-5′-diphospho-fucose, xanthosine-5′-diphospho-fucose,inosine-5′-diphospho-fucose, etc.⁷¹

Analogues of fucose activated as their diphosphate derivative which arerecognized by a particular fucosyltransferase so as to bind to theenzyme are then available for transfer to an appropriate acceptoroligosaccharide structure are said to be compatible with thefucosyltransferase. In so far as the fucose analogues are compatiblewith the fucosyltransferase, they are sometimes referred to herein as a“compatible fucose analogues”.

The term “oligosaccharide” refers to compounds of the formula

 R₁—R₂-(saccharide)_(n)-Y—R₃

wherein R₁ represents a saccharide residue, R₂ represents a saccharideresidue, and R₁ and R₂ together represent an acceptor for the selectedsialyltransferase and the selected fucosyltransferase; n is from 0 toabout 10, Y is selected from the group consisting of O, NH and S, and R₃is selected from the group consisting of H, a protein, a lipid or anaglycon moiety having at least one carbon atom.

The term “fucosylated oligosaccharide” refers to compounds of theformula

wherein R₁ represents a saccharide residue, R₂ represents a saccharideresidue, and R₁ and R₂ together represent an acceptor for the selectedsialyltransferase and the selected fucosyltransferase; n is from 0 toabout 10, Y is selected from the group consisting of O, NH and S, and R₃is selected from the group consisting of H, a protein, a lipid or anaglycon moiety having at least one carbon atom.

Since naturally occurring oligosaccharides and fucosylatedoligosaccharides are acceptors for certain α2,3-sialyltransferases, andare believed to be acceptors of certain sialyltransferases in vivo,these oligosaccharides and fucosylated oligosaccharides are sometimesreferred to herein as “natural acceptors”. Contrarily, since theoligosaccharides and fucosylated oligosaccharides employed in thisinvention are sometimes different from such “natural acceptors”, theyare sometimes referred to herein as “artificial acceptors”. That is tosay that artificial acceptors are those oligosaccharides and fucosylatedoligosaccharides which contain a substituent at the anomeric carbon atomof the reducing sugar which substituent is other than hydroxyl, aprotein, or a lipid capable of forming a micelle or other largemolecular weight aggregate. Accordingly, a protein linked to theanomeric carbon atom of the reducing sugar of the oligosaccharide orfucosylated oligosaccharide through its aglycon moiety would be anartificial acceptor since this acceptor contains an “artificial” unit,i.e., the aglycon linking group.

The fucosylated oligosaccharides of this invention may be furtherdistinguished from natural acceptors by virtue of chemicalmodification(s) to one or more of the saccharide units of theoligosaccharide. Such chemical modification could involve theintroduction and/or removal of one or more functionalities in one ormore of the saccharide unit(s). For example, such modification canresult in the removal of an OH functionality, the removal of saccharideunit(s), the introduction of an amine functionality, the introduction ofa halo functionality, the introduction of one or more saccharideunit(s), and the like.

In a preferred embodiment, the aglycon moiety has from 1-20 carbon atomsand, more preferably, is selected from the group consisting of —(A)—Z′wherein A represents a bond, an alkylene group of from 2 to 10 carbonatoms, and a moiety of the form —(CH₂CR₄R₅G)_(n)(CH₂CR₄R₅)— wherein n isan integer equal to 0 to 5; R₄ and R₅ are independently selected fromthe group consisting of hydrogen, phenyl, phenyl substituted with 1 to 3substituents selected from the group consisting of amine, hydroxyl,halogen, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4carbon atoms, methyl, or ethyl; and G is selected from the groupconsisting of a bond, oxygen, sulphur, NH, and Z′ is selected from thegroup consisting of hydrogen, methyl, —OH, —SH, —NH₂, —NHR₆, —N(R₆)₂,—C(O)OH, —C(O)OR₆, —C(O)NHNH₂, —C(O)NH₂, —C(O)NHR₆, —C(O)N(R₆)₂, and—OR₇ wherein each R₆ is independently alkyl of from 1 to 4 carbon atomsand R₇ is an alkenyl group of from 3 to 10 carbon atoms. Preferably, the—(A)—Z′ group defines a group capable of being linked to a carrier or agroup capable of being derivatized to a group which is capable of beinglinked to a carrier.

Preferably, the aglycon group is a hydrophobic group of at least 2carbon atoms and more preferably at least 4 carbon atoms. Mostpreferably the aglycon group is —(CH₂)₈COOMe.

When the aglycon group is one which is capable of being linked to acarrier such as an antigenic carrier, the methods of this invention areuseful in preparing artificial conjugates such as artificial antigenshaving one or more α-sialylated oligosaccharide groups containing ananalogue of sialic acid which groups are pendant to the antigen.

The carrier is a low or high molecular weight, nonimmunogenic orantigenic carrier including the linking to a fluorescent label, aradioactive label, biotin, or a photolabile linking arm or a moiety tobe targeted. Preferably, the carrier is an antigenic carrier andaccordingly, the artificial conjugate is an artificial antigen. In somecases it may be advantageous to employ a non-immunogenic carrier.

On the other hand, the carrier can be a low molecular weight carriersuch as ethylene diamine, hexamethylene diamine,tris(2-aminoethyl)amine, L lysilysine, poly-L-lysine, and polymers ofvarious molecular weights.

Saccharide units (i.e., sugars) useful in the oligosaccharides describedabove include by way of example, all natural and synthetic derivativesof glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine,fucose, sialic acid, 3-deoxy-D,L-octulosonic acid and the like. Inaddition to being in their pyranose form, all saccharide units in theoligosaccharides are in their D form except for fucose which is in its Lform.

As noted above, oligosaccharides useful in the processes disclosedherein contain terminal units which are compatible with the selectedsialyltransferase. That is to say that such compatible terminal unitspermit recognition of the oligosaccharide by a particularsialyltransferase so that the sialyltransferase binds to theoligosaccharide and further permits transfer of the compatible analogueof sialic acid onto the oligosaccharide.

B. Synthesis and Methodology

Preparation of Oligosaccharides

Oligosaccharides to which the sialic acid analogue is to beenzymatically coupled are readily prepared either by complete chemicalsynthesis or by chemical/enzymatic synthesis whereinglycosyltransferases (other than sialyltransferases) are employed toeffect the sequential addition of one or more sugar units onto asaccharide or an oligosaccharide. Such methods are well known in theart. For example, chemical synthesis is a convenient method forpreparing either the complete oligosaccharide glycoside; for chemicallymodifying a saccharide unit which can then be chemically orenzymatically coupled to an oligosaccharide glycoside; or for chemicallypreparing an oligosaccharide glycoside to which can be enzymaticallycoupled one or more saccharide units.

Chemical modifications of saccharide units are well known in the artwhich methods are generally adapted and optimized for each individualstructure to be synthesized. In general, the chemical synthesis of allor part of the oligosaccharide first involves formation of a glycosidiclinkage on the anomeric carbon atom of the reducing sugar. Specifically,an appropriately protected form of a naturally occurring or of achemically modified saccharide structure (the glycosyl donor) isselectively modified at the anomeric center of the reducing unit so asto introduce a leaving group comprising halides, trichloroacetimidate,thioglycoside, etc. The donor is then reacted under catalytic conditions(e.g., a soluble silver salt such as silver trifluoromethanesulfonate, aLewis acid such as boron trifluoride etherate ortrimethylsilyltrifluoromethanesulfonate, or thioglycoside promoters suchas methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfoniumtrifluoromethanesulfonate) with an aglycon or an appropriate form of acarbohydrate acceptor which possess one free hydroxyl group at theposition where the glycosidic linkage is to be established. A largevariety of aglycon moieties are known in the art and can be attachedwith the proper configuration to the anomeric center of the reducingunit. Appropriate use of compatible blocking groups, well known in theart of carbohydrate synthesis, will allow selective modification of thesynthesized structures or the further attachment of additional sugarunits or sugar blocks to the acceptor structures.

After formation of the glycosidic linkage, the oligosaccharide can beused to effect coupling of additional saccharide unit(s) or chemicallymodified at selected positions or, after conventional deprotection, usedin an enzymatic synthesis. In general, chemical coupling of a naturallyoccurring or chemically modified saccharide unit to the saccharideglycoside is accomplished by employing established chemistry welldocumented in the literature. See, for example, Okamoto et al.³²,Ratcliffe et al.³³, Abbas et al^(.34), Paulsen³⁵, Schmidt³⁶, Fugedi etal.³⁷, and Kameyama et al.^(.38). The disclosures of each of thesereferences are incorporated herein by reference in their entirety.

On the other hand, enzymatic coupling is accomplished by the use ofglycosyl transferases which transfer sugar units, activated as theirappropriate nucleotide donors, to specific saccharide or oligosaccharideacceptors, generally at the non-reducing sugar portion of the saccharideor oligosaccharide. See, for example, Toone et al.⁶² and U.S. Pat. No.5,374,655⁶⁷. Moreover, it is possible to effect selected chemicalmodifications of the saccharide or oligosaccharide acceptor, of thesugar donor or the product of the enzymatic reaction so as to introducemodifications or further modifications into the structure.

Preparation of Analogues of Sialic Acid

Certain analogues of sialic acid are well known in the art and areprepared by chemical modification of sialic acid using procedures welldocumented in the art. For example, chemically modified Neu5Acderivatives including 9-azido-Neu5Ac.³⁹, various 9-amino-Neu5Acderivatives⁴⁰, 9-deoxy-Neu5Ac⁴¹, 9-fluoro-Neu5Ac⁴², 9-bromo-Neu5Ac⁴³,8-deoxy-Neu5Ac⁴¹, 8-epi-Neu5Ac⁴⁴, 7-deoxy-Neu5Ac⁴⁷-epi-Neu5Ac⁴⁵,7,8-bis-epi-Neu5Ac^(.45), 4-O-methyl-Neu5Ac⁵³, 4-N-acetyl-Neu5Ac⁴⁸,4-epi-Neu5Ac⁴⁷, 4,7-di-deoxy-Neu5Ac⁴¹, 4-oxo-Neu5Ac⁴⁹, 4-deoxy-Neu5Ac⁵²,3-hydroxy-Neu5Ac⁵⁰, 3-fluoro-Neu5Ac⁵¹ acid, the product of cleavage ofthe side chain at C-8 or at C-7⁴⁶ as well as the 6-thio analogues ofNeu5Ac⁵⁴ are reported in the literature. Other sialic acid analogues aredisclosed in U.S. Pat. No. 5,352,670³. Chemical modification leading toother sialic acid analogues would follow such established procedures.

Activation of Analogues of Sialic Acid to Their CMP—NucleotideDerivatives

The enzymatic transfer of analogues of sialic acid require the priorsynthesis (i.e., activation) of their nucleotide (CMP) derivatives.Activation of the analogues of sialic acid is usually done by using theenzyme CMP-sialic acid synthase which is readily available and theliterature provides examples of the activation of various analogues ofsialic acid such as 9-substituted Neu5Ac^(28,39,40,55-57),7-epiNeu5Ac⁵⁸, 7,8-bis-epi-Neu5Ac⁵⁸, 4-O-methyl-Neu5Ac⁵⁹,4-deoxy-Neu5Ac⁶⁰, 4-acetamido-Neu5Ac⁴⁸, 7-deoxy-Neu5Ac⁵⁶,4,7-dideoxy-Neu5Ac⁵⁶, the 6-thio derivatives of Neu5Ac⁶¹ and Neu5OH(KDN). Still other examples of activated sialic acid analogues aredisclosed in U.S. Pat. No. 5,352,670³.

Transfer of the Analogues of Sialic Acid to the Oligosaccharide Acceptor

The nucleotide derivative of a compatible analogue of sialic acid andthe compatible acceptor (i.e., a fucosylated oligosaccharide or anoligosaccharide having terminal saccharide unit(s) on the non-reducingend which are recognized by the selected sialyltransferase) are combinedwith each other in the presence of the selected sialyltransferasecompatible with a fucosylated oligosaccharide under conditions whereinthe sialic acid or analogue thereof is transferred to the acceptor. Asis apparent, the saccharide or oligosaccharide acceptor employed must beone which functions as a substrate of the particular sialyltransferaseemployed.

In this regard, the art recognizes that artificial acceptors aretolerated in some cases by sialyltransferases especially wheremodification is in the aglycon part of the structure.

Likewise, when an analogue of sialic acid (i.e., an artificial donor) isto be enzymatically transferred, it is necessary that the CMP derivativeof the analogue also be recognized by the sialyltransferase. In thisregard, the art recognizes that certain sialyltransferases can toleratesome modifications to naturally occurring sialic acids and stilltransfer these analogues of sialic acid to glycoproteins or glycolipidspossessing a suitable terminal acceptor structure.

It has been found that sialyltransferases possess sufficient recognitionflexibility so as to transfer an artificial donor to an artificialacceptor³. Such flexibility permits the facile synthesis of a panel ofoligosaccharides containing different analogues of sialic acid at thenon-reducing sugar terminus of the oligosaccharide.

As noted above, a nucleotide derivative of a compatible sialic acid or acompatible analogue thereof is combined with a compatible acceptor(i.e., a saccharide or an oligosaccharide having terminal saccharideunit(s) on the nonreducing end which are recognized by the selectedsialyltransferase) in the presence of the sialyltransferase underconditions wherein the sialic acid or analogue thereof is transferred tothe acceptor. Suitable conditions, known in the art, include theaddition of the appropriate sialyltransferase to a mixture of thecompatible acceptor and of the CMP-derivative of the compatible sialicacid analogue in a appropriate buffer such as 0.1M sodium cacodylate inappropriate conditions of pH and temperature such as at a pH of 6.5 to7.5 and a temperature between 25° and 45° C., preferably 35-40° C. for12 hours to 4 days. The resulting oligosaccharide can be isolated andpurified using conventional methodology comprising HPLC, ion exchange-,gel-, reverse-phase- or adsorption chromatography.

Once formed, the α-sialylated oligosaccharide glycoside can be furthermodified by chemical and/or enzymatic means to further derivatize thiscompound. For example, other glycosyltransferases can be used to add aglycosyl group to an α-sialylated oligosaccharide recognized by thetransferase. This latter aspect is important insofar as themodifications made to the oligosaccharide must be compatible with thedesired enzymatic transfers.

Additionally, the α sialylated oligosaccharide can be chemicallymodified to provide further derivatization of these compounds. Suchchemical modification includes reduction of a 9-azido group on ananalogue of sialic acid to an amine group which can be still furtherfunctionalized to another derivative such as the 9-acetamido derivative.Similarly, the carboxyl group found on analogues of sialic acid can beselectively transformed on α sialylated oligosaccharide glycosides vialactonization, reduction or transformation into an amide.

In one or more of the enzymatic steps recited above, the enzyme can bebound to a solid support so as to facilitate the reaction of thereagents and the recovery of the product from the enzyme.

C. Utility

The methods of this invention are useful in preparing oligosaccharidescontaining sialic acid or an analogue thereof bound via an α-linkage tothe non-reducing sugar terminus of the oligosaccharide. Sucholigosaccharides are recognized in the art as being useful aspharmaceuticals, as well as in the generation of antibodies to thesestructures, which antibodies are useful in diagnostic assays.

Additionally, methods of this invention are useful in preparingoligosaccharides containing an analogue of sialic acid bound via anα-linkage to the non-reducing sugar terminus of the oligosaccharidewhich can be coupled to an antigenic carrier so as to produce artificialantigens. Accordingly, such oligosaccharides act as intermediates in thepreparation of artificial antigens.

Additionally, methods of this invention are useful in the determinationof the non-reducing terminus of an unknown oligosaccharide.Specifically, a first aliquot of the unknown oligosaccharide acceptorcan be contacted with a sialyltransferase which is not capable ofsialylating a non-reducing terminus of an oligosaccharide having afucose group in the non-reducing penultimate saccharide position anddetermining whether the oligosaccharide was sialylated; a second aliquotof the unknown oligosaccharide is also contected with an α-2,3sialyltransferase which is capable of sialylating a non-reducingterminus of an oligosacchairde having a fucose group in the non-reducingpenultimate saccharide position and determining whether theoligosaccharide was sialylated; the results are compared to determinewhether the non-reducing terminus was fucosylated. If the first aliquotof the unknown acceptor was not sialylated and the second aliquot ofacceptor was sialylated then the acceptor was fucosylated in thenon-reducing penultimate saccharide position.

EXAMPLES

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of this invention.

In these examples, unless otherwise defined below, the abbreviationsemployed have their generally accepted meaning:

PBS = phosphate buffered saline MES = morpholine ethane sulfonic acidPMSF = α-toluenesulfonyl fluoride Le^(x)-gr = 8-methoxycarbonyloctylα-L-fucopyranosyl- (1→3)-[β-D-galactopyranosyl-(1→4)]-β-D-2-acetamide-2-deoxy-glucopyranoside Le^(a)-gr = 8-methoxycarbonyloctylβ-D- galactopyranosyl-(1→3) [α-L-fucopyranosyl-(1→4)]-β-D-2-acetamide-2-deoxy- glucopyranoside CMP-NANA = cytidine5′-monophospho-N-acetyl-neuraminic acid CMP-³H-NANA = cytidine5′-monophospho-N-acetyl-neuraminic acid [sialic-9-³H] BSA = bovine serumalbumin d = doublet dd = doublet of doublets s = singlet t = tripletGDP-Fuc = guanosine-5′-diphospho-L-fucose UDP-galactose =uridine-5′-diphospho-galactose TMR = tetramethylrhodamine

Commercially avaliable components are listed by manufacturer. Some ofthe recited manufacturers are as follows:

Millipore=Millipore Corp., Bedford Mass.

Waters=Waters Corp., Milford, Mass.

Boehringer Mannheim=Boehringer Mannheim, Laval, Quebec, Canada

Example 1

Preparation of Viral α2,3-sialyltransferase Cell Lysates

The myxoma viral α2,3-sialyl transferase cell lysate was prepared by amethod similar to that set forth in International Patent ApplicationPublication No. WO97/18302⁶⁵, which is incorporated herein by reference.

Ten T180 flasks of confluent layers of European rabbit kidney cell(RK13) cells were infected with Brazilian myxoma virus strain, Lausanne(Lu) (ATCC VR-115) isolated Campinas, Brazil, 1949 and Uriarra (Ur)isolated Australian Capital Territory, 1953 (a derivative of Mosesstrain (ATCC VR-116)). The cells were kept at 37° C. for 24 hours.Twenty-four hours post infection, the cells were detached by scrapingand washed with PBS. Cell lysates were prepared by suspension in 20 mLof extraction buffer (50 mM MES, pH 6.1, 0.5% Triton-X100, 100 mM NaCl,1.5 mM MgCl₂, 0.1 mM PMSF, 10 mg/ml aprotinin) at 4° C., for 45 minutes.The lysate was clarified by centrifugation at 2,000 g at 4° C. for 15minutes.

The supernatant was recovered and applied to a 5 mL HiTrap Blue Affinitychromatography column (Pharmacia, Piscataway N.J.) in loading buffer (50mM MES, pH6,1, 0.1% Triton-CF54, 100 mM NaCl, 25% glycerol). Theα2,3-sialyltransferase was eluted from the column with a step NaClelution (0.5 M, 1.0 M, 1.5 M, and 2.0 M NaCl). Theα2,3-sialyltransferase was desalted by passing the eluant through aPD-10 column (BioRad, Hercules, Calif.) in column buffer (50 mM MES, pH6,1, 0.1% Triton-CF54, 25% glycerol).

Total protein concentrations were measured using Bradford Bio-Rad andfollowing the manufacturers instructions with IgG as a protein standard.

Example 2

Transfer of Sialic Acid to Lewis^(a) and Lewis^(x) ligosaccharideAcceptors

Acceptor (54 nmol), CMP-NANA (40 nmol), and CMP-³H-NANA (150,000-180,000dpm) were added to a mixture of cell lysate (16 μL), water (3 μL) and 1μL of assay buffer (250 mM MES, 0.5% Triton CF54, pH 7.0) in a 0.5 mLmicrofuge tube. Reaction mixtures were incubated at 37° C., diluted withwater to 200 μL and loaded onto a C₁₈ Sep-Pak reverse-phase cartridgewhich had been pre-equilibrated with 20 mL of MeOH and 20 mL of water.The cartridge was washed with 50 mL of water and the product eluted with4 mL of MeOH into a scintillation vial. The radioactivity of the MeOHeluates were quantitated by liquid scintillation in 10 mL of EcoLite (+)scintillation cocktail (ICN, Montreal, Quebec, Canada) in a Beckmanliquid scintillation counter (LS1801).

Results of the radioactive transfer to the different acceptors induplicate experiments were as follows:

Acceptor total CMP-NANA Incubation time dpm Le^(x)-gr 152644  90 min.1330/1130 Le^(a)-gr 176174 170 min. 4161/4045

Example 3

Transfer of Sialic Acid to Various Oligosaccharide Acceptors

The ability of the isolated viral α2,3-sialyltransferase to transfer asialic acid to various acceptors was tested.

Acceptor (54 nmol), CMP-NANA (40 nmol), and CMP-³H-NANA (150,000-180,000dpm) were added to a mixture of cell lysate (16 μL), water (3 μL) andassay buffer (250 mM MES, 0.5% Triton CF54, pH 6.1) in a 0.5 mLmicrofuge tube. Reaction mixtures were incubated at 37° C., diluted withwater and measured by the method set forth in Example 2 above to obtainrelative rates of transfer. Kinetics were carried out in an analogousmethod by varying the acceptor concentration from about 0.2×Km to 3×Km.

Relative Rate Relative rVmax/ Acceptor (2.7 mM) Km (μM) Vmax KmLacNAc—O-gr 100 112 ± 11 100 (1.5 0.896 nmol/ mL/ min) 6′,6′-C-dimethyl-17 LacNAc-O-octyl¹ Lactose-O-gr 90 211 ± 40 112 0.5313′-C-methyl-lactose- 4.1 octyl¹ 4′-C-methyl-lactose- 20 octyl¹Le^(c)-O-gr 79 202 ± 10 90 0.446 T-disaccharide-O-gr 64  427 ± 110 510.119 Galα(1→3)-lacNAc-O-gr 18 Galα(1→4)-lactose-O-gr 12  9270 ± 1848 170.00183 GlcNAc-O-gr <1 Glc-O-octyl <1 Gal-O-phenyl <1 Fuc-Gal-O-octyl1.5 LacNAc-OH 171 Lactose-OH 121  97 ± 43 120 1.23 Le^(a)-O-gr¹ 50 1578± 105 42 0.0266 Le^(x)-O-gr¹ 21  9490 ± 1930 30 0.00316 CMP-NANA 244 ±36 7-LacNAc-O-gr¹ 1.4 gr = (CH₂)_(8 COOMe) CMP-NANA = cytidine5′-monophospho-N-acetyl-neuraminic acid ¹These compounds serve asacceptors for the viral α-2,3 sialyltransferase but are not acceptorsfor previously known mammalian α-2,3 sialyltransferase.

This indicates that the viral α2,3-sialyltransferase is able to use anumber of different oligosaccharide structures as acceptors for thetransfer of a sialic acid.

Example 4

Confirmation of 2,3 Linkage of Sialic Acid to Lewis_(a) and Lewis_(x)oligosaccharide Acceptors

Le^(a)-TMR (35 nmol) and CMP-NANA (200 nmol) were incubated with viralcell lysate (4.9 μL) and alkaline phosphatase solution (0.1 μL) (5 μL ofalkaline phosphatase (Boehringer Mannheim) 1000 U/ml and 1 μL BSAsolution (100 mg/mL)). After gentle rotation at room temperature (25°C.) for 42 hours, additional alkaline phosphatase solution (0.2 μL) andCMP-NANA solution (100 mM, 0.2 μL) were added to the mixture which wasreacted for 2 more days at room temperature. The reaction mixture waselevated and maintained at 37° C. for 48 hours, then the mixture wasloaded onto a C₁₈-Sep-Pak cartridge (Waters) which had beenpre-equilibrated with 10 mL of MeOH and 10 mL of water. The cartridgewas washed with 5 mL of water then TMR-labeled compounds were elutedwith 5 mL of MeOH. This solution was dried under vacuum, passed througha filter (Milliex-GV filter, 0.22 μm, Millipore Corp.) and lyophilized.Water was added to dry material to make 100 μM TMR concentration. Thissolution (0.5 μL) was mixed with capillary electrophoresis runningbuffer (10 mM phosphate, 10 mM sodium borate, 10 mM sodium dodecylsulfate, 10 mM phenyl boronic acid pH 9.0, 499.5 μL). This solution wasused for separation and analysis by capillary electrophoresis with laserinduced fluorescence detection by the method set forth in Le et al.⁶⁶ Anew product peak produced in the enzyme reaction had the same migrationtime as authentic sialylated Le^(x)-TMR and the new product peak wasconverted back to Le^(x)-TMR by treatment with neuraminidase.

Example 5

Preparative Synthesis of SialylLe^(x)-gr

Cell lysates (14 mL) were mixed with BSA solution (5 μL, 100 mg/mL).This solution was concentrated in a Slide-A lyzer (Pierce ChemicalCompany, Rockford, Ill.) to 1.8 mL. Le^(x)-gr (4.7 mg, 6.7 μmol) andCMP-NANA (6.8 mg, 10.3 μmol) were added to 200 μL of concentrated celllysate Alkaline phosphatase (Boehringer Mannheim, 1000 U/mL, 10 μL) wasadded. This reaction mixture was turned at room temperature for 24 days.During this incubation, CMP-NANA was added (6 times, after 3 days, 7days, 11 days, 3.0 mg each, after 14 days, 18 days, 21 days, 2.0 mgeach). This mixture was loaded onto a C₁₈-Sep-Pak Pak cartridge (Waters)which had been pre-equilibrated with 10 mL of MeOH and 10 mL of water.The cartridge was washed with 40 mL of water, then the product waseluted with 50 mL of 10% MeOH. This eluate was dried under vacuum andagain loaded onto a C₁₈-Sep-Pak cartridge which had beenpre-equilibrated with 10 mL of MeOH and 10 mL of water. After washingwith water (10 mL); 1% MeOH (10 mL), and then 5% MeOH (10 mL), thedesired product was eluted with 10% MeOH (17 mL). This solution wasdried under vacuum, passed through a filter (Milliex-GV filter, 0.22 μm,Millipore Corp.) and lyophilized to give sialylated Le^(x)-gr (2.19 mg,33%). The structure of the product sialyl Le^(x)-gr was confirmed byboth NMR spectroscopy and mass spectrometry.

NMR (300 Hz, only typical peaks are shown, D₂O) δ5.10 (d, H, J=3.9 Hz,H-1(Fuc)), 4.51 (d, 2H, J=8.0 Hz, H-1(Gal, GlcNAc)), 3.69 (s, 3H,CO₂Me), 2.76 (dd, H, J=4.7, 12.6 Hz, H-3, (NANA)), 2.38 (t, 2H, J=11.4Hz, CH₂CO₂Me), 2.04 (s,3H, Ac), 2.02 (s, 3H, Ac), 1.79 (t, H, J=12.2,1.8 Hz, H-3 (NANA)), 1.17 (d, 3H, J=6.6 Hz, H-6 (Fuc)).

Mass calculated for C₄₁H₆₉N₂O₂₅=989.4; found 989.0

Sialylated Le^(a)-gr was also synthesized from Le^(a)-gr in a similarmanner. Its structure was confirmed by both NMR spectroscopy and massspectrometry.

NMR (300 Hz, only typical peaks are shown, D₂O) δ5.00 (d, H, J=3.9 Hz,H-1(Fuc)), 4.52 (d, 2H, J=7.7 Hz, H-1(Gal, GlcNAc)), 3.69 (s, 3H,CO₂Me), 2.76 (dd, H, J=4.7, 12.5 Hz, H-3, (NANA)), 2.39 (t, 2H, J=7.3Hz, CH₂CO₂Me), 2.02 (s, 6H, Ac), 1.76 (t, H, J=12.3, H-3 (NANA)), 1.16(d, 3H, J=6.4 Hz, H-6 (Fuc)).

Mass calculated for C₄₁H₆₉N₂O₂₅=989.4; found 989.0

Example 6

Synthesis of SialylLe^(x)-TMR from GlcNAc-TMR

GlcNAc-TMR (35 nmol), UDP-galactose (70 nmol), 0.5 μL isolated bovinemilk β-1,4 galactosyltransferase (0.3 mU), GDP-fucose (70 nmol), 0.5 μLisolated human milk α1,3,4-fucosyltransferase (0.03 mU), CMP-NANA (100nmol), 0.1 μL of 1 M MnCl₂ and 0.1 μL alkaline phosphatase (BoebringerMannheim, 10 mU) were incubated with 5.7 μL of concentrated viral celllysate solution. After gentle rotation at room temperature for 24 hours,0.2 μL aliquots were removed and spotted onto a silica gel 60F₂₅₄ thinlayer chromatography plate (Merck, Darmstadt Germany). The plate wasdeveloped with isopropanol:H₂O:NH₄OH (7:2:1). Le^(x)-TMR (Rf=0.18) andsialyl Le^(x)-TMR (Rf=0.30) were visible due to the pink chromophoreTMR. These Rfs correspond to those of authentic material and the sialylLe^(x)-TMR formed in the enzyme reaction mixture co-migrated with sialylLe^(x)-TMR. The starting material GlcNAc-TMR (Rf=0.39) and the reactionintermediate LacNAc-TMR (Rf=0.25) were not detected.

Example 7

Synthesis of SialylLe^(a)-TMR from Le^(c)-TMR

Le^(c)-TMR (15 nmol), GDP-fucose (70 nmol), 0.5 μL isolated human milk,α-1,3,4-fucosyltransferase (0.03 mU), CMP-NANA (100 nmol), 0.1 μL of 1 MMnCl₂ and 0.1 μL alkaline phosphatase (Boehringer Mannheim, 10 mU) wereincubated with 5.1 μL of concentrated viral cell lysate solution. Aftergentle rotation at room temperature for 72 hours, 0.2 μL aliquots wereremoved and spotted onto a silica gel 60F₂₅₄ thin layer chromatographyplate (Merck, Darmstadt Germany). The plate was developed withisopropanol:H₂O:NH₄OH (7:2:1). Le^(a)-TMR (Rf=0.18) and sialylLe^(a)-TMR (Rf=0.25) were visible due to the pink chromophore TMR. TheseRfs correspond to those of authentic material and the sialyl Le^(a)-TMRformed in the enzyme reaction mixture co-migrated with sialylLe^(a)-TMR. The starting material Le^(c)-TMR (Rf=0.31) was not detected.

What is claimed is:
 1. A method for determining the non-reducingterminus structure of an unknown oligosaccharide acceptor, which methodcomprises the steps of: a) contacting the oligosaccharide acceptor witha sialyltransferase which is not capable of sialylating a non-reducingterminus of an oligosaccharide having a fucose group in the non-reducingpenultimate saccharide position and determining whether theoligosaccharide was sialylated; b) contacting the oligosaccharide withan α-2,3 sialyltransferase which is capable of sialylating anon-reducing terminus of an oligosacchairde having a fucose group in thenon-reducing penultimate saccharide position and determining whether theoligosaccharide was sialylated; and c) comparing the results todetermine whether the non-reducing terminus was fucosylated.
 2. Themethod according to claim 1, wherein step (c) comprises determining thatthe unknown acceptor was fucosylated in the non-reducing penultimatesaccharide position where the acceptor was not sialylated in step (a) ofclaim 1 and was sialylated step (b) of claim 1.